M2 protein in the influenza A membrane coat is a known drug target. Unfortunately, the S31N mutation has caused widespread drug resistance in H3N2 since 2006 and in the recent H1N1 swine flu pandemic. Excellent progress has been made in understanding the H+ transport activity of this protein through characterizations of truncated versions of M2 that form partially functional transporters. At the heart of this tetrameric protein are four histidine residues that have been shown to have unique chemical properties responsible for the acid activation of the pore and suspected of being directly involved in the transporter mechanism for H+ conductance. Drug binding in the pore dramatically influences the pKas of the histidine residues, displaces waters that facilitate helix kinking, and induces the closure of the Val27 secondary gate. Studies of the M2 truncates have been complicated by membrane mimetic environments used for structural characterization. The comparison of structures obtained by x-ray crystallography, solution NMR and solid state NMR spectroscopy clearly show that the environment has a substantial influence upon secondary, tertiary, and quaternary levels of protein structure. Here, structural, dynamical, and functional characterizations of the full-length protein will all be performed in bilayers having a lipid composition similar to that of the viral membrane. Liposome and oocyte assays of H+ conductance, magic angle spinning and aligned sample solid state NMR spectroscopy, and molecular dynamics simulations will be performed using nearly identical environments for the full-length M2 protein. High-resolution complete (backbone and sidechains) structural characterizations as a function of pH and in the presence and absence of drug will be achieved. This structural data will be coupled with conductance assays and molecular dynamics simulations to achieve a quantitative atomistic mechanism for selective proton conductance and acid activation. Similarly, data from the S31N mutant and in the presence and absence of amantadine will be combined to reach a detailed understanding of how the drug blocks conductance in the wild-type protein but not in the S31N mutant. Detailed functional understanding at this level is difficult to achieve for a membrane protein. Here, a proven interdisciplinary team will meet this challenge for a proven drug target that could once again be an important factor in the defense against a deadly influenza pandemic. PUBLIC HEALTH RELEVANCE: Influenza is a serious health threat in the US and around the world and the M2 protein of this virus represents an essential function making it one of the few proven drug targets for this virus. While we have recently solved the structure of the conductance domain in a lipid bilayer environment, here we propose to characterize the full length protein in a lipid bilayer modeling the viral coat with a team having expertise in conductance assays, molecular dynamics simulations and quantum mechanics - molecular mechanics calculations, and solid state NMR spectroscopy. The aim for this research is to develop for the M2 protein mechanistic understanding of its selective proton conductance, acid activation, conductance blockage by drugs, and drug resistance.